Cells react to microorganismsby activating innate immune responses that relyon recognitionof non-self molecular patterns, such as the bacterial cell wall component lipopolysaccharide (LPS). Exposure of the adherent neutrophil to the LPS component of gram negative bacteria results in an integrated response involvingactivation of JNK MAP kinase, which is involved in a variety of downstream functional effectsthat may contribute tothe pathogenesis of ARDS. We propose that the adherent neutrophilassembles a signaling complex involving activation of tyrosine kinase Syk, and utilizesadaptor molecules, particularly SLP-76, previously known in mediating signal transduction in the lymphocyte, but never previously implicated in neutrophilsignal transduction. We further suggest that the JNK pathway is regulated not only in the activation of membrane- proximal adaptors, but is also influenced by cross-talk between p38 MAP kinase and JNK, We suggest that p38 activates PP2A, a phosphatase that inactivates MKK4. Using a variety of techniques to detect molecular interactions and modify expression levels in human and murine neutrophils, and transfectable neutrophilic cell lineswe will address 2 major specific aims: 1. To define the molecular mechanisms by which the LPS receptor complex activates JNK in the adherent neutrophil.2. To determine the mechanisms by which p38 regulates activationof the JNK pathway. The results of these investigationswill highlightnovel aspects of neutrophil signal transduction in response to LPS that lead to activation of JNK, and provide insights intothe assembly of multi-component signaling complexes whose interactionswill provide new approaches to modulation. PERFORMANCESITE(S) (organization,city, state) NationalJewishMedicaland ResearchCenter, Denver,Colorado KEYPERSONNEL. See instructions. Use continuationpagesas needed to providethe required informationin the format shown below. Startwith Principal Investigator.List all other key personnel inalphabetical order,last namefirst. Name Organization Role on Project Worthen, G. Scott National Jewish Medical and Research Center P.I. Duncan, Mark University of Colorado Health Sciences Center Consultant Johnson, Gary University of Colorado Health Sciences Center Consultant Koretzky, Gary University of Pennsylvania Consultant Lieber, Jonathan National Jewish Medical and Research Center Research Fellow Malcolm, Kenneth C. National Jewish Medical and Research Center Research Fellow Brian Wadzinsky, Ph.D. Vanderbilt University Medical Center Consultant Disclosure Permission Statement. Applicableto SBIR/STTROnly. See instructions. [] Yes [] No + PHS 398 (Rev.05/01) Page 2 FormPage 2 + ! Principal Investigator/Program Director (Last, first, middle): Worthen, G. Scott The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page ......................................................................................................................................................... 1 Description,